EP2501475B1 - System and a method for detecting analyte molecules contained in liquid samples - Google Patents

Description

The invention relates to a system and a method for detecting analyte molecules contained in liquid samples. These may in particular be proteins or DNA. Particularly advantageous use is possible with very small molecules

Usually, the procedure is such that a liquid sample flows through a measuring channel in which ligands specific for the respective analyte molecules are immobilized on measuring surfaces to which the analyte molecules can bind. After binding, a detection is carried out in which it can be determined whether the respective analyte molecules in the sample are included or not. A quantitative determination can also be made.

The analyte molecules are more or less evenly distributed in the liquid sample and the measurement channel has a certain required volume. As a result, the liquid sample flows through the measuring channel with a minimum layer thickness. However, a complete filling of measuring channels is preferred. The transport of analyte molecules to the immobilized ligands takes place essentially by convection and diffusion. Near the surface of measurement surfaces where ligands are immobilized, a layer is formed in which diffusion substantially occurs. This is called the Nernst-type diffusion barrier layer. The transport of analyte molecules to ligands is thereby hindered, whereby this effect increases with increasing thickness of the diffusion layer.

To counteract these disadvantages and to increase the binding rate of analyte molecules and accelerate the binding was in DE 10 2007 012 866 A1 proposed to guide through a flow channel a main stream formed with an inert liquid. In these main streams free of analyte molecules, a feed for liquid sample should then be arranged in front of the actual measuring surfaces. The main flow can be used to displace the liquid sample in the direction of the measuring surfaces with the ligands immobilized there. The liquid sample can thus flow as a thin film over the measuring surfaces.

It is obvious that due to the required larger free cross-section of the flow channel an enlargement of the entire system. With the main stream, a dilution effect for the sample can not be avoided. In addition, the binding behavior of particular analyte molecules can not be influenced in a specific or selective manner.

Furthermore, it is known that a separation or sorting of nanoparticles or biomolecules is possible by means of dielectrophoresis. A suitable device is in the unpublished DE 10 2008 062 620 described.

In this case, strip-shaped electrodes should be arranged at intervals above one another and optionally also below a measuring channel at intervals from one another and in each case acted upon by an electrical alternating voltage. The polarity changes from electrode to electrode. In this case, with a certain frequency taking into account the respective Clausius-Mossotti factor, a force action is to be exerted on molecules in order to move them away when passing through them to a measuring surface or non-specifically bound foreign molecules.

In this case, differences in the liquid in which the respective analyte molecules are contained, and it is very complicated to determine the respective the Clausius-Mossotti factor considering frequency. Even if the frequency of the alternating electrical voltage is at least optimally maintained, the analyte molecules must have a certain size in order to achieve a sufficient force effect.

Out DE 697 29 101 T2 For example, a device and a method for separating, immobilizing and quantifying biological substances are known. This should be done with help be achieved by external and internal magnetic fields, which are to be formed with permanent magnets, which are arranged with respect to a ferromagnetic catcher construction on a separation vessel. The catcher construction should preferably be formed with a grid formed of a ferromagnetic material, which is to be attached to the upper inner wall of the separation vessel. The force effect of the magnetic field is intended to draw molecules or cells which are coupled with magnetically reacting particles in the direction of the capture structure and to immobilize there. Subsequently, a preferred optical detection of the molecules can then be carried out.

In particular, by the arrangement of the catcher construction but a detection is hindered also unspecifically bound molecules affect the accuracy and also a cleaning can not be easily performed or in a simple form.

FR 2 863 117 - A1 describes a microsystem for displacing fluids, the microsystem having a micro-conduit with at least one fluid and means used to establish a primary displacement of the fluid between an inlet and an outlet of the micro-conduit. Furthermore, the microsystem has magnetohydrodynamic means which cause at least a secondary displacement of the fluid. The invention can be used in the field of microfluidics, inter alia for mixing fluids and for scanning particles on the surface of reactors.

It is therefore an object of the invention possibilities to achieve improved sensitivity of analyte molecules, with a simple design and reusable system.

According to the invention, this object is achieved with a system having the features of claim 1. It can proceed with a method according to claim 5. Advantageous embodiments and further developments of the invention can be realized with features designated in subordinate claims.

In the system according to the invention, in each case at least one opening for supplying and discharging thereof is present in a housing in the flow direction of a fluid containing analyte molecules at a measuring channel at its beginning and end. A sample comprising analyte molecules and a liquid can thus be passed through the measurement channel. In addition, at the bottom of the measuring channel there is a sensitive area on which ligands for the respective analyte molecules can be immobilized in the measuring channel.

The housing should be formed of a non-magnetic and non-magnetizable material. For this purpose, suitable polymers and / or aluminum can be used.

Above the measuring channel at least one element made of a ferromagnetic material is arranged in the housing material or on the upper wall of the measuring channel. For the external formation of a magnetic field, two permanent magnets are arranged on both sides of the measuring channel parallel to the flow direction, or they can be arranged there temporarily. A magnetic field should be within the measurement channel at least in the region in which the / the element (s) is arranged / are made of ferromagnetic material, are formed. In this case, the analyte molecules have a susceptibility> 0 or particles are bound to analayt molecules whose susceptibility is> 0. In the case of particles bound to analyte molecules, the total susceptibility should be> 0. The analyte molecules and / or particles therefore have paramagnetic, supermagnetic or ferromagnetic properties.

In a system not belonging to the invention, a plurality of permanent magnets may be arranged in a series arrangement above the measuring channel. The permanent magnets are alternately magnetized alternately. The polar alignment of juxtaposed permanent magnets is therefore set against.

This series arrangement should be arranged at least in the region of the sensitive surface area.

For detection, in this system not belonging to the invention, a sample is used in which analyte molecules are present which have a susceptibility which is ≦ 0 or particles which have a susceptibility ≦ 0 are bound to analyte molecules. In the case of particles bound to analyte molecules, the total susceptibility should be ≤0. The analyte molecules and / or particles have diamagnetic properties.

When flowing through the sample comes in both alternative training in the sphere of influence of the magnetic Field, which is formed with the permanent magnets, so that the analyte molecules are acted upon by a force acting in the direction of the bottom of the measuring channel and the immobilized with ligands for analyte molecules sensitive surface area.

It can be exploited here that a force acting on magnetic or magnetized particles can be influenced by the gradient of the magnetic field strength within a magnetic field. The respective force is dependent on the ratio of the susceptibility of the particles and the surrounding medium.

In the invention, it can be utilized that a magnetic field formed with magnets can be influenced with ferromagnetic elements which are arranged in the magnetic field. These elements are magnetized and can lead to magnetic field intensity gradients in certain directions, depending on the particular arrangement of the magnets and the ferromagnetic elements. In the plane aligned parallel to the external magnetic field, the gradient becomes a ferromagnetic element. Perpendicular to this, the gradient of the magnetic field strength away from the ferromagnetic element.

The direction-dependent force F is given by $$\mathrm{F}={\mathrm{½V}}_{\mathrm{P}}*\left({\mathrm{\chi }}_{\mathrm{p}}-{\mathrm{\chi }}_{\mathrm{fl}}\right)*{\mathrm{\mu }}_{\mathrm{0}}*\mathrm{\delta }\left(\mathrm{H}*\mathrm{H}\right)/\mathrm{\delta }\mathrm{,}$$

Where χ _{p is} the susceptibility (magentizability) of the analyte molecules or of the magnetizable particles / particles bound to them, χ _fl - the acceptability of the sample liquid in each case as dimensionless volume magnetizability in SI unit, Vp volume of the analyte molecules optionally with bound magnetisable particle and H - the magnetic field strength.

By a suitable choice can accordingly the term (χp - χ _fl) be positive or negative, whereby the direction of the acting force F can be changed by 180 ° in accordance depending on the sign. A suitable parameter for this is a corresponding selection of a liquid for the respective sample with a smaller or larger susceptibility χ _fl . as the susceptibility χp of the analyte molecules.

One or more elements of ferromagnetic material should preferably be arranged in the flow direction in front of the sensitive surface area. Thereby, a force acting on the analyte molecules, which in this case have a higher susceptibility χ _p as the susceptibility χ _fl of the sample liquid from the one or more ferromagnetic element (s) away in towards the bottom of the measuring channel and because of the flow in the direction ligands immobilized on the sensitive area are exercised. The respective analyte molecules can thereby be better and more securely bound to the ligands. The required sample volume can be kept small and the required time can be reduced.

The ferromagnetic elements which can be used in the invention can have a very flat design and can be aligned parallel to the bottom of the measuring channel. They may, for example, be aligned in a strip shape and parallel to the flow direction of the sample. They should have a thickness of 0.1 mm to 0.4 mm have their width at least ten times greater.

It may also be preferable to use only such a planar ferromagnetic element. Its width should be at least 80% of the width of the measuring channel in the flow direction.

One or more elements of ferromagnetic material can be conveniently embedded in the material of the housing. Direct contact with samples can thus be avoided. It can maintain a permanent exact positioning and avoid adhesion problems. These elements can also be formed in the form of wires with a circular or elliptical cross-section.

With the two or the permanent magnets, a magnetic field with a magnetic field strength H of at least 0.5 T should be able to be formed. The magnetization of the permanent magnets should be at least 0.5T.

As already indicated above, by selecting a suitable liquid with corresponding greater susceptibility χ _fl, the direction of force action which is caused by the gradient of the magnetic field strength can also be changed with an unchanged arrangement of the permanent magnets. This can be used for rinsing or also removing / detaching unspecifically bound other molecules from the sensitive surface area. Instead of a sample, before or even after detection, a liquid with greater susceptibility χ _fl , in which no further molecules, at least no analyte molecules contained, flow through the measuring channel. Unspecifically bound molecules whose binding forces to ligands are smaller can be easily detached and removed from the measuring channel before the actual detection of the analyte molecules.

At a significantly greater susceptibility χ _fl such a liquid may also be achieved a complete flushing of the measuring channel and thereby at least the bound ligand analyte molecules are removed so that a so-purged and purified system is used for detection of another sample again.

For example, whole blood samples, blood plasma or other body fluids may be used for detection. Such samples may also be more or less diluted. This can be achieved with deionized water.

For rinsing, it is possible to use, for example, manganese (II) chloride or gadolinium (III) complexes as liquid with greater susceptibility χ _fl .

To improve the magnetizability very small ferromagnetic, paramagnetic or superparamagnetic particles can be bound to the respective analyte molecules whose size is a few nanometers. These particles can be formed from iron, nickel, cobalt or an alloy of said metals or they can also be used as a mixture with polymer.

However, the same effects for rinsing and dissolving non-specifically bound molecules can also be achieved by adjusting the orientation of the external magnetic field is changed. In this case, the previously arranged on the two sides of the measuring channel two permanent magnets can be removed. At least one permanent magnet is then arranged above the one or more elements formed from a ferromagnetic material. The element (s) are then located between this magnet and the measuring channel. The force caused by the gradient of the magnetic field strength thereby changes its direction and is opposite to the direction of force, which has been exploited for binding the analyte molecules to ligands. The magnitude of the acting force is dependent on the achievable with the / the permanent magnet magnetic field strength and / or its / their distance to the / the ferromagnetic element (s).

A third possibility for purging and / or removing nonspecifically bound molecules consists of flowing through the measuring channel with a rinsing liquid in the opposite direction through the measuring channel.

The abovementioned possibilities for rinsing and / or removing unspecifically bound molecules can also be used combined with one another.

The invention will be explained in more detail by way of example in the following.

• Figur 1 auf magnetisierbare Teilchen in einem magnetischen Feld wirkende Kraftvektoren, die bei der Erfindung ausgenutzt werden können;
• Figur 2 eine schematische Darstellung eines erfindungsgemäßen Systems für ein Ablenken von Analytmolekülen in Richtung auf einen sensitiven Flächenbereich am Boden eines Messkanals;
• Figur 3 eine schematische Darstellung eines Systems für ein Ablenken von Analytmolekülen in die entgegen gesetzte Richtung vom Boden eines Messkanals weg, das nicht zur Erfindung gehört;
• Figur 4 eine Explosionsdarstellung eines Beispiels eines erfindungsgemäßen Systems, bei dem zwei Permanentmagnete an den Seiten eines Messkanals angeordnet werden können und
• Figur 5 eine Explosionsdarstellung eines weiteren Beispiels eines erfindungsgemäßen Systems.

Showing:

• FIG. 1 force vectors acting on magnetizable particles in a magnetic field which can be exploited in the invention;
• FIG. 2 a schematic representation of a system according to the invention for deflecting analyte molecules towards a sensitive area at the bottom of a measuring channel;
• FIG. 3 a schematic representation of a system for deflecting analyte molecules in the opposite direction from the bottom of a measuring channel, which does not belong to the invention;
• FIG. 4 an exploded view of an example of a system according to the invention, in which two permanent magnets can be arranged on the sides of a measuring channel and
• FIG. 5 an exploded view of another example of a system according to the invention.

With FIG. 1 It should be clarified how the direction of forces acting on magnetic or magnetizable particles in a magnetic field is dependent on the orientation of the magnetic field. This can be changed by changing the orientation of the magnetic field.

In the schematic representation after FIG. 2 is shown in a side and front view formed in a housing 1 of optically transparent polymer measuring channel 3 through which a sample 2 is guided. The flow direction is indicated by the arrow. Particles of iron, nickel, an alloy thereof, which can also be used as a mixture with polymer, having a diameter of 5 nm to 500 nm are bound to the respective analyte molecules of sample 2. The thus-prepared analyte molecules had a susceptibility χ _P > 0 to 100. The Liquid of the sample had a susceptibility χ _fl <0. When using liquids with different Suszeptibilät χ _fl , for improved attachment of analyte molecules, the particles may also be magnetizable polymers or a diamagnetic metal, such as gold. The improvement of the attachment by liquids with increased susceptibility leads to a negative difference in the term (χ _P - χ _fl ) to a deflection of particles or analyte molecules which are diamagnetic or bound to the diamagnetic particles. However, this only applies to the second alternative of the invention.

In the lid 1.1 of the housing 1, a ferromagnetic element 6 formed of iron is embedded in the polymeric material. The element 6 has a thickness of 0.2 mm. Its width should be 2.5 mm. The measuring channel 3 has a length of 8 mm to 10 mm with respect to the flow direction and a height of 50 microns.

On the two sides of the measuring channel 3, not shown here permanent magnets 5 are mounted, with the aid of an external magnetic field can be formed. In this case, the north pole of a permanent magnet 5 points in the direction of the measuring channel 3, whereas the south pole of the permanent magnet 5 arranged on the opposite side of the measuring channel 3 faces the measuring channel 3. The magnetization of the permanent magnets 5 should be at least 0.5 T.

With FIG. 2 is illustrated by the darker area of the flowing through the measuring channel 3 Sample 2, as analyte molecules move through the forces caused by the gradient of the magnetic field strength towards the bottom of the measuring channel 3. There, a sensitive area 7 is formed, are immobilized on the ligand for analyte molecules. The sensitive area 7 may be formed with a thin metal layer, preferably gold or silver. In this case, it is possible to perform the detection by means of an SPR analysis, as for example in DE 10 2008 062 620 is described. For this purpose, an unillustrated optical waveguide can be arranged below the sensitive surface region 7, via which electromagnetic radiation can be directed at least almost under total reflection conditions onto the underside of the sensitive surface region 7. The evaluation of the SPR analysis can be carried out in a manner known per se.

With FIG. 3 a system not belonging to the invention is shown in schematic form. Here, 3 permanent magnets 5.1 and 5.2 are arranged in the flow direction of the sample 2 in series above the measuring channel. The juxtaposed permanent magnets 5.1 and 5.2 are each magnetized opposite to each other. The susceptibility χ _{p of} the analyte molecules or possibly also of the particles which are bound to analyte molecules is smaller than the susceptibility χ _{fl of} the liquid or the fluid of the sample 2. It becomes clear that as in the example of FIG. 2 a force is exerted on the analyte molecules in the sample 2 and can be utilized for a movement in the direction of the sensitive area 7 arranged at the bottom of the measuring channel 3 in order to improve the binding behavior of the analyte molecules to ligands immobilized there.

For rinsing or removal of unspecifically bound molecules, in which FIG. 2 example shown, instead of the sample only a rinsing liquid with a higher susceptibility χ _fl than the susceptibility of the sample liquid can be used. The sign of the term (χ _p - χ _fl ) changes and as a result the direction of the acting forces changes in the opposite direction, which can lead to the detachment of nonspecifically bound molecules. With a larger difference of χ _p and χ _fl , all molecules can be detached and the measuring channel 3 can be cleaned.

An example of a system according to the invention used for binding analyte molecules to ligands is disclosed in US Pat FIG. 4 shown in an exploded view. In this case, two permanent magnets 5 can be inserted laterally to the right and left of the measuring channel 3 into recesses 9 arranged there or can be used temporarily for a detection. Above the measuring channel 3, a plate-shaped element 6 made of iron is embedded in the lid 1.1 of the housing 1 in the polymer of the lid 1.1. The element 6 has the following dimensions L / B / H 10 / 2.5 / 0.2 mm.

As already described, a sensitive surface area 7 is again formed at the bottom of the measuring channel 3.

When flowing through a sample 2, as already in the description of FIG. 2 explained, act on the magnetized analyte molecules forces that accelerate them towards the bottom of the measuring channel 3 and the sensitive area 7 and there can be achieved in the area above the sensitive surface area 7 an enrichment of analyte molecules, thereby improving the bonding behavior and increases the binding rate can be.

Between the lid 1.1 and bottom 1.3 of the housing 1, a sealing element 1.2 is disposed of an elastomer, pointing to the bottom in the direction of the bottom 1.3 facing a recess is present, which forms the measuring channel 3. On the surface of the bottom 1.3 is a sensitive area 7, formed as a thin gold layer. There, ligands can be immobilized.

In the lid 1.1, a further receptacle 10 is formed, in which a further permanent magnet 8 can be used when bound analyte molecules or non-specifically bound molecules to be removed. The permanent magnets 5 previously inserted into the receptacles 9 have been removed therefrom.

The direction of the acting forces has been reversed by the use and the arrangement of the permanent magnet 8. It now points away from the bottom 1.3 of the measuring channel 3 in the direction of the element 6 formed from ferromagnetic material. Thus, detachment and removal of molecules, including those bound to ligands, from the measuring channel 3 can be achieved.

The openings for the supply and removal of samples 2 may be formed in the lid 1.1.

The FIG. 5 shows a system according to the invention without the two arranged on the sides of the measuring channel 3 permanent magnets. 5
Otherwise this corresponds to in FIG. 5 shown system by example FIG. 4 , wherein, however, can be additionally dispensed with the formation of shots 9 and 10 in the lid 1.1.

Claims (9)

1. A system for the detection of analyte molecules contained in liquid samples in which at least one respective opening for the supply and removal of a sample (2) containing analyte molecules is provided at a start and at an end of a measurement channel (3) within a housing (1) in the direction of flow of said sample containing analyte molecules;
   in which in addition a sensitive surface region (7) is arranged at the base (1.3) of the measurement channel (3); and
   in which the housing (1) is formed from a nonmagnetic and non-magnetisable material,
   characterised in that
   at least one element (6) composed of a ferromagnetic material is arranged above the measurement channel (3) in the housing material or at the upper wall of the measurement channel (3); and in that in addition two permanent magnets (5) are arranged at both sides of the measurement channel (3) in parallel with the direction of flow, with which permanent magnets a magnetic field is formed within the measurement channel (3) at least in that region in which the element(s) (6) of ferromagnetic material is/are arranged and in this respect analyte molecules having a susceptibility > 0 or particles whose susceptibility > 0 and are bound to analyte molecules are contained in the sample (2) so that the analyte molecules can be acted on by a force acting in the direction toward the sensitive surface region (7) immobilised by ligands for analyte molecules.
2. A system in accordance with claim 1, characterised in that the element(s) (6) of ferromagnetic material is/are arranged before the sensitive surface region in the direction of flow.
3. A system in accordance with claim 1 or claim 2, characterised in that the ferromagnetic element(s) has/have a thickness in the range from 0.1 mm to 0.4 mm and its/their width(s) is/are at least ten times larger.
4. A system in accordance with one of the preceding claims, characterised in that an element (6) of a ferromagnetic material whose width amounts to at least 80% of the width of the measurement channel (3) in the direction of flow is arranged within the housing (1) above the measurement channel (3).
5. A method for the detection of analyte molecules contained in liquid samples, in which a sample containing analyte molecules is conducted through a measurement channel (3), which is configured within a housing (1) formed from nonmagnetic or non-magnetisable material, via a sensitive surface region (7) which is arranged at the base of the measurement channel (3) and on which ligands for the respective analyte molecules are immobilized;
   on flowing through the measurement channel (3), the sample (2) enters into the region of influence of an externally formed magnetic field which is formed by two permanent magnets (5) arranged at both sides of the measurement channel (3) and which is formed by means of at least one element (6) of ferromagnetic material arranged above the measurement channel;
   such that a gradient of the magnetic field strength occurs within the measurement channel (3) which results in a force effect in the direction of the measurement channel (3) and of the sensitive surface region (7) toward the analyte molecules such that they are accelerated in the direction of ligands and are bound thereto, wherein
   in the magnetic field formed by two permanent magnets (5), the analyte molecules have a susceptibility > 0 or particles whose susceptibility is > 0 are bound to analyte molecules.
6. A method in accordance with claim 5, characterised in that permanent magnets (5) are used having a magnetisation of at least 0.5 T.
7. A method in accordance with claim 5 or claim 6, characterised in that a flushing liquid having an opposite direction of flow or a liquid having a higher susceptibility χ_fl than the susceptibility χ_p of the analyte molecules is conducted through the measurement channel (3) for a flushing and/or for a removal of non-specifically bound molecules, with simultaneously permanent magnets (5) arranged at both sides of the measurement channel (3) and with an unchanged magnetic field.
8. A method in accordance with claim 5 or claim 6, characterised in that a flushing liquid is conducted through the measurement channel (3) for the removal of non-specifically bound molecules and in this connection the two permanent magnets (5) at the sides are removed and at least one permanent magnet (8) is arranged above the measurement channel (3) and above the element(s) (6) of ferromagnetic material.
9. A method in accordance with any one of the claims 5 to 7, characterised in that to increase the susceptibility of χ_p of the analyte molecules the latter are bound to ferromagnetic, paramagnetic or superparamagnetic particles before the introduction of the sample into the measurement channel (3).

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